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1 http://journals.tubitak.gov.tr/agriculture/ Turkish Journal of Agriculture and Forestry urk J Agric For(2014) 38: © ÜBİAKdoi:10.3906/tar-1306-47 Efficient in vitro plant regeneration from immature embryos of endemic Iris sari and Iris schachtii Satı UZUN 1, *, Ali İrfan İLBAŞ 2 , Arif İPEK 3 , Neşet ARSLAN 4 , Surendra BARPETE 4 1 Department o Field Crops, Faculty o Agriculture, Erciyes University, Kayseri, urkey 2 Department o Agronomy, Faculty o Agriculture, Kyrgyzstan-urkey Manas University, Bishkek, Kyrgyzstan 3 Department o Biology, Faculty o Science, Çankırı Karatekin University, Çankırı, urkey 4 Department o Field Crops, Faculty o Agriculture, Ankara University, Ankara, urkey * Correspondence:
[email protected] 1. Introduction Environmental pressures such as agriculture, erosion, overgrazing o meadows, and urbanization cause significant loss o plant species and their habitats. Tereore, special attention should be paid to protect and preserve the endemic flora that is normally ound in limited areas (Fay, 1992; Sarasan et al., 2006). Conservation o endemic species by in vitro plant tissue culture has attracted the attention o researchers (Mallon et al., 2010; Piovan et al., 2010; Corral et al., 2011). In vitro techniques make the rapid and reliable production o a large amount o individuals rom a small amount o plant material possible, and thus are preerred as an efficient way or conservation o endemic plants and wild populations (Corral et al., 2011). In addition, they play a major role in the production o plant material required or genetic transormation, virus elimination, rejuvenation o mature plant material, and the acquisition o industrial raw material (Endress, 1994; Hasançebi et al., 2011).Te genus Iris belongs to the amily Iridaceae, which contains 43 species in urkey, 13 o which are endemic (Aslan and Karataş, 2012). Iris sari and Iris schachtii are presented in the ‘least concern’ category in the Red Data Book o urkish Plants (Ekim et al., 2000). Both species have potential as ornamental plants with their attractive flowers. A recent trend o using wild plants or ornamental purposes by collecting them rom their natural habitats could lead to the extinction o wild plants (Prado et al., 2010). Iris species can be propagated rom seeds, bulbs, or rhizomes with a low propagation speed like other geophytes (Jevremovic and Radojevic, 2002; Boltenkov et al., 2007). Tereore, in recent years, a number o studies have reported in vitro propagation o Iris species such as I. ensata (Yabuya et al., 1991; Boltenkov et al., 2007), I. germanica (Shimizu et al., 1997; Wang et al., 1999a, 1999b), I. nigricans (Shibli and Ajlouni, 2000), I. pumila (Jevremovic and Radojevic, 2002), I. ensata , I. setosa , I. sanguinea (Boltenkov and Zarebno, 2005), I. hollandica (Fidalgo et al., 2005), I. atrofusca , I. petrana , I. vartanii (Al-Gabbiesh et al., 2006), and I. adriatica (Keresa et al., 2009) rom a range o explants.In the present study, rapid and reliable protocols or in vitro shoot regeneration, rooting, and acclimatization o endemic I. sari and I. schachtii were developed. Te study presents the first in vitro shoot regeneration o I. sari and I. schachtii , which can be valuable or commercial production and germplasm conservation o both endemic species. Abstract: Plant tissue culture is an efficient technique or conserving endemic plant species. A reproducible in vitro regeneration protocol was developed or the endemic Iris sari and Iris schachtii in the present study. Te highest number o shoots per explant was obtained on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L thidiazuron (DZ) plus 0.5 mg/L α-naphthaleneacetic acid (NAA) and 1 mg/L DZ plus 0.5 mg/L NAA, whereby 96.88% and 100% shoot induction with 9.55 and 11.34 shoots per explant o Iris sari and Iris schachtii were recorded, respectively. Regenerated shoots were successully rooted on MS medium with either 1 mg/L indole-3-butyric acid (IBA) or 1 mg/L IBA plus 0.2 mg/L NAA. Rooted shoots were transerred to pots containing either a peat-soil-sand (1:1:1) mixture or a hydroponic culture containing Hoagland solution to acclimatize the regenerated plants to the greenhouse chamber. Approximately 90% o plants were transerred ex vitro successully. Key words: Endemic species, immature embryo, in vitro regeneration, Iris sari , Iris schachtii Received: 15.06.2013 Accepted: 12.09.2013 Published Online: 00.00.2013 Printed: 00.00.2013 Research Article 2 UZUN et al. / Turk J Agric For 2. Materials and methods 2.1. Surface disinfection of plant materials I. sari and I. schachtii plants were collected rom the Erzurum and Ankara provinces o urkey and planted in the experimental fields o the Agricultural Faculty at Ankara University, Ankara (urkey). Immature pods containing immature zygotic embryos approximately 1 mm in length were harvested, washed with running tap water, and then surace sterilized with 50% commercial bleach [5% (v/v) sodium hypochloride, ACE, urkey] or 10 min, washed 3 times with sterile distilled water or 5 min each, and blotted onto tissue paper. 2.2. Shoot regeneration from zygotic immature embryos Immature pods were dissected under sterile conditions and the seed was squeezed using orceps until the immature zygotic embryo was released. Immature embryos were then placed onto Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962) supplemented with 3% (w/v) sucrose, 0.7% plant agar (w/v, Duchea, the Netherlands), 6-benzylaminopurine (BAP; 1, 2, or 4 mg/L), thidiazuron (DZ; 0.5 or 1 mg/L), or kinetin (KIN; 2 or 4 mg/L), with or without 0.5 mg/L α-naphthaleneacetic acid (NAA). Explants were placed in 100 × 10 mm petri dishes containing 25 mL o culture medium and incubated in the growth chamber. Cultures were transerred to resh media at 4-week intervals. Each treatment contained 6 explants or I. schachtii and 8 explants or I. sari , with 4 replicates. Te requency o shoot regeneration and mean number o shoots per explant was determined afer 12 weeks. 2.3. Rooting of the regenerated shoots In vitro regenerated shoots that srcinated rom immature embryos were rooted on 1 mg/L indole-3-butyric acid (IBA) alone or in combinations with 0.2 or 0.4 mg/L NAA. Plant growth regulator (PGR)-ree MS medium was also used as control. For rooting experiments, 10 regenerated shoots were used and each treatment was replicated 4 times. Data on root induction ratio, mean number o roots per shoot, and mean root length were recorded afer 2 months o culture.Te pH levels o all media were adjusted to 5.6–5.8 beore autoclaving or 20 min at 121 °C. All cultures were grown at 24 ± 2 °C with a 16-h photoperiod. 2.4. Acclimatization For acclimatization, healthy plantlets with well-developed root and shoot systems were chosen. Tey were removed rom the culture media, washed in running tap water to remove agar, and then transerred to plastic pots containing either sterile soil-sand-peat (1:1:1) or hydroponic culture containing Hoagland solution. Each vessel or pot was covered with a transparent polyethylene bag to create high relative humidity; aferwards, the bags were gradually opened by perorating small holes in the bag. Afer 10 days the bags were opened completely. ransplanting plants were maintained in the growth chamber at 24 ± 2 °C with a 16-h photoperiod. 2.5. Statistical analysis Te data were evaluated by analysis o variance (ANOVA) and the differences between means were compared by Duncan’s multiple range tests. Data given in percentages were subjected to arcsine (√X) transormation (Snedecor and Cochran, 1967) beore statistical analysis. 3. Results 3.1. Shoot regeneration from zygotic immature embryos In the present study, shoot regeneration response was observed in all culture media irrespective o MS medium supplemented with or without NAA in I. sari and I. schachtii (able 1). Morphogenic green callus appeared rom the explants afer 2 weeks and development o shoots was observed afer 4–5 weeks o culture (Figure 1a), ollowed by well-developed shoots afer 12 weeks (Figures 1b and 1c).Significant variations in the requency o shoot regeneration were determined depending on PGRs (P < 0.01). Te requency o shoot regeneration ranged rom 78.12%–100% and 70.84%–100% in I. sari and I. schachtii , respectively (able 1). Te highest shoot requency was obtained rom all media except 1 mg/L DZ, 2 mg/L KIN, and 4 mg/L KIN. Inclusion o NAA used with BAP, DZ, or KIN resulted in increased shoot regeneration requency compared to use alone. Mean number o shoots per explant varied significantly depending on species, PGRs, and the interactions between species and PGRs (P < 0.01). Te highest number (9.55) o shoots per explant in I. sari was achieved on MS medium supplemented with 0.5 mg/L DZ plus 0.5 mg/L NAA. Te maximum o 11.34 shoots per explant o I. schachtii was observed on medium with 1 mg/L DZ plus 0.5 mg/L NAA. Comparing effects o PGRs, DZ proved to be more efficient than BAP and KIN on shoot multiplication in I. sari. On the other hand, DZ and BAP produced more shoots than KIN in I. schachtii (able 1). Te addition o 0.5 mg/L NAA with all concentrations o BAP, DZ, and KIN exerted positive effects on shoot prolieration in both species. However, the combination o DZ plus NAA promoted a considerable increase in the number o shoots per explant relative to that induced by combinations o BAP plus NAA and KIN plus NAA in both species. 3.2 Rooting of the regenerated shoots and acclimatization Root development was observed on all rooting mediums with or without auxins; most o the shoots were rooted within 2 months (Figures 1d and 1e). While rooting percentage and mean root length were significantly affected by PGRs (P < 0.01), mean number o roots per shoot was 3 UZUN et al. / Turk J Agric For affected by PGRs and interaction (species and PGRs, P < 0.05). Te lowest rooting requency was recorded on MS medium devoid o auxins in both species (52.50%). Root inductions o I. sari and I. schachtii (95% and 92.5%, respectively) were promoted with 1 mg/L IBA, with mean root lengths observed as 7.10 and 5.88 cm, respectively (able 2). Maximum mean number o roots per shoot o I. sari (4.37) and I. schachtii (4.80) was recorded on MS medium supplemented with 1 mg/L IBA and 1 mg/L IBA plus 0.2 mg/L NAA, respectively. Te presence o 0.4 mg/L NAA with IBA resulted in decreased rooting requency and mean root length. Plantlets with well-developed roots were transerred to either plastic pots containing a 1:1:1 mixture o peat-soil-sand (Figures 2a and 2b) or hydroponic culture containing Hoagland solution (Figures 2c and 2d) and kept in a growth chamber under a 16-h light photoperiod. Afer 3 months, the survival rates o I. sari and I. schachtii were 56% and 61% in peat-sand-soil mixture and 89.5% and 91.89% in hydroponic culture, respectively (data not shown). 4. Discussion Efficient shoot regeneration has been previously reported rom immature embryos o geophytes (Mirici et al., 2005; Uranbey, 2010; Nasırcılar et al., 2011). Immature embryos are suitable explants or in vitro shoot regeneration and they may prevent overharvesting o the parent plant rom natural populations. Te cytokinins BAP and KIN, and cytokinin-like substance DZ (alone or in combination with auxin), are commonly used to induce shoot regeneration in plant tissues (Özcan et al., 1996; Çöçü et al., 2004; Aasim et al. 2013) and especially to promote organogenesis rom immature explants (Özcan et al., 1992; Özcan et al., 1996; Mirici et al., 2005; Uranbey 2010). According to the results o the current study with I. sari and I. schachtii , immature embryo explants showed better response when cultured on MS medium supplemented with DZ plus NAA. Te MS medium with 0.5 mg/L DZ plus 0.5 mg/L NAA and 1 mg/L DZ plus 0.5 mg/L NAA produced the largest number o regenerated shoots per explant. Although DZ has been proven to be effective or in vitro regeneration o various plants (Çöçü et al., 2004; Erişen et al., 2011; Nasırcılar et al., 2011), Shibli and Ajlouni (2000) reported that embryogenesis did not occur with DZ treatment in Iris nigricans . Te lowest shoot regenerations were obtained rom 2 mg/L or 4 mg/L KIN-containing media or both species, Table 1. Effect o plant growth regulators (PGRs) on shoot regeneration o I. sari and I. schachtii. PGRs (mg/L)Frequency o shoot regeneration (%)Mean number o shoots per explantBAPNAA I. sariI. schachtii Mean I. sariI. schachtii Mean1-87.50100.0093.75 ab*3.55 g*7.63 de*5.59 g*10.5100.0100.00100.00 a5.58 bcde9.58 bc7.58 bc2-81.2595.888.54 ab3.75 eg8.54 cd6.15 de 20.596.8895.8396.35 ab5.63 bcd8.79 bcd7.21 cd4-81.25100.0090.63 ab4.00 deg9.63 abc6.81 cde40.5100.087.5093.75 ab5.25 cde9.17 bcd7.21 cdDZ0.5-93.7595.8394.79 ab7.05 b9.96 abc8.50 b0.50.596.8895.8396.35 ab9.55 a10.45 ab10.00 a1-87.5087.5087.50 bc4.70 cdeg5.75 g5.23 gh10.596.87100.0098.44 ab5.85 bc11.34 a8.59 bKIN2-78.1270.8474.48 c4.00 deg4.42 g4.21 h20.584.3891.6788.02 ab4.00 deg5.37 g4.69 gh4-90.6283.3486.98 bc3.25 g4.83 g4.04 h40.593.7587.5090.62 ab5.00 cdeg6.33 e5.67 egMean*90.6392.26--5.08 b7.99 a-- *: Values within a column or row ollowed by different letters are significantly different at the 0.05 probability level o Duncan’s multiple range test. 4 UZUN et al. / Turk J Agric For in line with the findings o Shimizu et al. (1997), who reported that KIN had an inhibitory effect on somatic embryogenesis but no effect on shoot development in I. germanica . On the other hand, KIN was generally used in the induction o embryogenic callus in irises (Gozu et al., 1993; Shimizu et al., 1996; Wang et al., 1999a, 1999b). Tis variation may stem rom genotype and concentration or combination o PGRs. Boltenkov and Zarembo (2005) reported that plant species and PGRs play an important role in the establishment o regeneration. In treatments including BAP, shoot regeneration response changed with species and I. schachtii was ound to be better than I. sari . Te promotive effect o BAP alone or in combination with NAA-inducing callus and adventitious bulblet regeneration was previously reported in other Iris species (Kawase et al., 1995; Suzhen et al., 1999; Shibli and Ajlouni, 2000; Boltenkov and Zarembo, 2005; Boltenkov et al., 2007).Te effectiveness o MS basal medium supplemented with 1 mg/L IBA was demonstrated or better root development with 95% and 92.5% successul rooting in I. sari and I. schachtii , respectively. As observed by Laublin et al. (1991), IBA-containing medium allowed rapid development o roots in Iris species. Hal-strength MS Figure 1. In vitro propagation and rooting o I. sari and I. schachtii: (a) shoot initiations rom callus afer 1 month o culture; (b) multiple shoots ormation I. sari and (c) I. schachtii afer 3 months o culture; (d ) root ormation o I. sari and (e) I. schachtii . Table 2. Effects o IBA and NAA combinations on in vitro rooting o I. sari and I. schachtii .PGRs (mg/L) Root induction (%)Mean number o roots per shootMean root length (cm)IBANAA I. sariI. schachtii Mean I. sariI. schachtii Mean I. sariI. schachtii Mean--52.5052.5052.50 c*3.29 a*2.00 b*2.65 b*4.444.684.56 c*1-95.0092.5093.75 a4.37 a3.45 a3.91 a7.105.886.49 a10.282.5092.5087.50 a3.96 a4.80 a4.38 a6.105.545.82 ab10.477.5077.5077.50 b3.14 a4.29 a3.72 a5.164.434.79 bcMean78.8878.75--3.693.64--5.705.13--- *: Values within a column followed by different letters are significantly different at the 0.05 probability level of Duncan’s multiple range test. 5 UZUN et al. / Turk J Agric For plus 0.5 mg/L IBA were reported to have better impacts on rooting growth o Iris lactea Pall. var. chinensis (Meng et al., 2009). However, with shoots cultured in medium without PGRs, rooting success was only 52.5% in both species. Boltenkov et al. (2007) suggested that PGRs were required or root initiation in Iris ensata . Acclimatization is a critical stage and ofen associated with slow growth and significant plant loss. Wang et al. (1999b) stated that plant survival and growth afer transer rom in vitro culture to ex vitro were affected by potting substrate in I. germanica . In the present study, hydroponic culture yielded better survival rates than a 1:1:1 peat-soil-sand mixture. Similarly, Zapata et al. (2003) observed the best growth, as well as the maximum survival percentage o plants acclimatized in the hydroponic system, in Curcuma longa L. In conclusion, an efficient and rapid in vitro regeneration protocol was developed or I. sari and I. schachtii , ollowed by high requency o rooting and acclimatization protocols. Tese results could be useul or the propagation and conservation o these endemic species. Acknowledgment Tis work was supported by the Scientific Research Foundation o Erciyes University, Project Number FBA-10-3206. Figure 2. Acclimatization o in vitro regenerated plantlets: (a) I. sari , (b) I. schachtii , (c and d) hydroponic culture o regenerants. References Aasim M, Day S, Rezaei F, Hajyzadeh M (2013). Multiple shoots regeneration o plumular apices o chickpea. urk J Agric For 37: 33–39. Al-Gabbiesh A, Hassawi DS, Afifi FU (2006). In vitro propagation o endangered Iris species. J Biol Sci 6: 1035–1040.Aslan S, Karataş A (2012). Iris tuberosa var. longifolia ( Iridaceae ) üzerine sistematik notlar ve yeni bir yayılış alanı. In: Proceedings o the 21st Ulusal Biyoloji Kongresi, September, 2012; İzmir, urkey (in urkish). Boltenkov EV, Mironova N, Zarembo EV (2007). Effect o phytohormones on plant regeneration in callus culture o Iris ensata Tunb. Biology Bulletin 34: 446–450.Boltenkov EV, Zarembo V (2005). 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